Influence of phosphate on the allosteric behavior of yeast phosphofructokinase

Vol. 78, No. 4, 1977

BIOCHEMICAL

INFLUENCE BEHAVIOR

Michel

OF

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PHOSPHATE

OF

YEAST

ON THE

ALLOSTERIC

PHOSPHOFRUCTOKINASE

LAURENT and Fransois

SEYDOUX

Laboratoire d'Enzymologie Physico-Chimique Universitg de Paris-Sud, 91405, Orsay

Received

et Moleculaire - France.

June 23,197l

SUMMARY Initial rate data obtained with purified yeast phosphofructokinase (PFK) show an ATP dependent kinetic cooperativity with respect to fructose-6-phosphate. In the presence of 25 mM phosphate, the cooperativity index (Hill number) is related to the half saturation concentration of fructose-6-phosphate as predicted by the concerted allosteric model in the case of a "K-system". In the absence of phosphate, however, the kinetic behavior of yeast PF'K is more complex and the cooperativity index is invariant with respect to the half saturation concentration of fructoseE-phosphate which is increased by ATP. In both cases, 5'AMP behaves as a strong activator of the enzyme. These kinetic data suggest that the two distinct functions of ATP as phosphate donnor and as allosteric inhibitor, are supported by different binding sites. These regulatory respectively, properties of yeast PFK are discussed in relation to glycolytic oscillations.

INTRODUCTION The regulatory various

species

properties

have been extensively

PFKd is one of the key enzymes also,

from

losteric with

other

regulation acellular

been proposed models

share

tiated at the for glycolytic x Abbreviation

Copyright All rights

independant

the occurence

and yeast

of phosphofructokinase

studies,

(3-5).

These

extracts for

(7). these

during

the past

the glycolytic an oligomeric

regulatory

of biochemical

to account

studied

controlling

in

Numerous

kinetic

remarkable that

ten years (I,21

El 1977 by Academic Press, Inc. of reproduction in any form reserved.

(I).

and is

complex

yeast

cells

interpretations

al-

Most

(6) have

of these

oscillations

site of PFK (a-10). The development of a quantitative oscillations requires, however, a precise knowledge : PFK , phosphofructokinase

from

have been correlated

intact

phenomena.

glycolytic

flux

enzyme with

properties

oscillations

in common the postulate

isolated

are

ini-

model of the

(EC 2.7.1.11)

1289 ISSN

0006-291X

Vol. 78, No. 4, 1977

kinetic

behavior

of the currently analyze yeast

the effect

BlOCHEMlCAL

of yeast available

AND BlOPHYSlCAl

PFK and its

quantitative

allosteric

of phosphate

models

on the

RESEARCH COMMUNICATIONS

description

(IL).In

steady

state

in terms

the present kinetics

paper,

we

of purified

PFK.

EXPERIMENTAL PFK has been purified from commercial baker's yeast (Springer, France) according to the procedure described by Diezel et al. (12) with minor modifications. The enzyme preparation showed less than 5% of proteolytic degradation as estimated from polyacrylamide gel electrophoresis in 0.1% SDS (12).Phosphoenolpyruvate, ATP, 5'AMP and NADH were purchased from Sigma. Fructose-6-phosphate, fructose 1,6-diphosphatase and other coupling enzymes were obtained from Boehringer. All other chemicals were of the best available purity. A coupled assay with pyruvate kinase (5 units/ml) and lactate dehydrogenase (5 units/ml) was used for the determination of PFK activity. For experiments performed without S'AMP, 0.1 unit/ml of fructose 1,6diphosphatase was added to the reaction mixture. This regenerating system avoids the accumulation of products and allows accurate measurements of initial velocities. The reaction was started by addition of the enzyme (final concentration 0.6 ug/ml) diluted in 10 mM phosphate buffer (pH 6.8) containing 1 mM dithiothreitol, 1 mM MgC12, 1 mM EDTA and 1 mM fructose6-phosphate. All kinetic measurements were carried out at 25OC, pH 6.8 in 50 mM Tris-sulfonate buffer with 1 mM dithiothreitol, 5 mM NH4C1, 1 mM phosphoenolpyruvate and 0.2 mM NADH. In all cases, a three 5 mM M&12, fold molar excess of MgC12 with respect to ATP was maintained. Under these conditions, phosphoenolpyruvate, KC1 and MgC12 have no detectable effect on the enzyme activity. Initial rate data were analysed according to the empirical Hill equation: nH v m . CS) v= (eq. 1)

K”H 0.5

dH

+

the velocity extrapolated at inwhere v is the observed reaction rate, V finite concentration of fructose 6-phosphate (S), n the Hill number and KC the half maximum rate concentration of fructose 6-p Eosphate. Data were * fitted in the range of 10 to 90 % of saturation to eq. 1 using an iteration procedure similar to that described by Atkins (13).

RESULTS The rate dance

with

the kinetic and without saturation

respect

of the

PFK catalyzed

to fructose

reaction

6-phosphate

exhibits

concentration.

a sigmoidal Fig.

depen-

1 and 2 show

saturation curves obtained at various ATP concentrations with added phosphate, respectively. In the presence of phosphate, the curves

are

well

described

over

1290

the entire

range

of saturation

by

Vol. 78, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

6-

4-

Z, Is

-1.4

-0.2

-0.8

0.4

0

1-OG(FRU-6-P)

Fig.

1 :

Allosteric behavior of yeast PFK in the presence of 25 mM K2HP04: Initial rates as function of the fructose 6-phosphate concentration (mN). Experimental conditions : ( v ) 240 PM ATP t 1 mM 5'AMP, ( LJ ) 12 UM ATP, ( 0 ) 79 PM ATP, ( 0 ) 119 LIM ATP, ( + ) 238 NM ATP, ( n ) 1.19 mM ATP, i 0 ) 4.69 mM ATP. Other conditions as described in the experimental section. Rates are normalized with respect to the maximum velocities which are given in fig.3. Solid curves correspond to the best fit of the data to the Hill equation (eq. 1). The insert shows the relationship between the Hill number and the half saturation concentration K for fructose 6$osphate . The dashed line is calculated P.5 rom the equation: =

nH

1

+

(Ko 5 - $,,) (Ko', t KR)

(n-1)

(KR - c.Ko 5) (KP + c.K 0.5)

where n is the apparent number of protomers of the functional oligomer, c is the non-exclusive binding coefficient for fructose 6-phosphate and K is the apparent dissociation constant of fructose 6-phosphate for t Re R state (14). The following values of the parameters were used :n= 3, c = 0.020 and KR = 52 !JM.

the

empirical

the half

Hill

te increase

with

dependance

respect the

on VW . As shown respect

The values

(KO 5) with

3. Addition values

in fig. basis

in the

of the maximum rate respect

The VW parameter

to fructose exhibits

(Km for

of S'AMP to the reaction

(VW) and 6-phospha-

a Michaelian

ATP = 3i LJM) mixture

decrea-

of K any significant effect 0.5 and n H without dependance of the observed Hill number nH

1, the

to the value on the

a "K system"

1).

to the AT? concentration

in fig.

ses significantly with

(eq.

concentration ATP concentrations.

with

as illustrated

described

equation

saturation

of the half

saturation

of the bellshaped

concerted

allosteric

model

1291

parameter

relationship (3,151.

(14)

KO 5 is which

adequatly

characterize

Vol. 78, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

LOG
Fi :g. 2:

Allosteric behavior of yeast PFK in the absence of phosphate. Experimental conditions : ( v ) 760 PM ATP + 1 mM 5'AMP, ( n ) 12 PM ATP, ( 0 ) 79 ~.IM ATP, ( 0 ) 160 UM ATP, ( n ) 790 yM ATP, ( 0 ) 4 mM ATP. Other conditions as in fig. 1 . The insert shows the invariance of the Hill number nH with respect to the half saturation concentration K0.5 in this case.

'.&,/IS1 [UNITS/ Fig.

3 :

different, velocities

/Nl

Michaelian saturation of yeast PFK by ATP. Eddie plot of the reaction rates extrapolated at infinite fructose 6-phosphate concentration as function of the ATP concentration (S). Maximum rates were calculated from the data of fig. 1 ( v , 25 ml? phosphate) and fig. 2 ( m , no phosphate).

In the absence various

MG I

however.

As shown

ATP concentrations obey the

of phosphate, in fig.

are roughly

same Michaelian

the

situation

appears

2, the saturation parallel. relationship

1292

to be quite

curves

Although as in the

obtained

at

the maximum presence

of

Vol.

78, No.

4, 1977

the apparent

phosphate, two

(see

tions

BIOCHEMICAL

fig.

3).

Km for

In addition,

show considerable

ration

range,

phdSe

of the

werful

that

saturation of the

respect

to the half

behavior

is clearly

not

RESEARCH

COMMUNICATIONS

by a factor

the rate

at low

data

obtained

from the

high

Hill

equation

cooperativity

curves.

Although

reaction,

the

saturation

BIOPHYSICAL

ATP (16 yM) is smaller

deviation

indicating

activator

AND

parameter

Kg

with

that

consistent

only

still

number

n

ATP roncentr~i-

in the

occurs

5'AMP is Hill

O-20% satll-

in the

in this is

predicted

for

initial

case a po-

invariant

witn

H shown in fig.

5 as

elf ,~bout

2. This

a "K system".

DISCUSSION The role

of phosphate

has been described results only

previously

obtained

with

as a classical

the

qualitative

a purified

of the

in the

presence

data

(16).

phosphate

also

is

that

PFK contains

yeast

functions

in

not

fully

to the nH versus

!i) ficantly

the

ATP acts

(ii) is

for

likely

of phosphate in these

significantly

Our results

V_ with

site

which

is

that

this increase

interaction

also

cannot

of its

may have distinct suggest

that

ATP

:

does not displace by the

signi-

Michaelian

dcpendance

behavior

for

this

ATP and fructose observed

1293

binding

of yeast site,

6-phosphate, Phosphate

6-phosphate

of AT? as illustrated in the presence for

equidecreases

fructose substrate.

the peculiar

sites

PFK in the

the conformational

co-substrate

however,

inhibitor.

by distinct

to the active

the binding

account,

consi-

be remembered

reaction

supported

KC, 5 value

Km value

is

has been found the allosteric

as an allosteric

are

between with

the

to ATP.

upon binding

affinity

of the

which

and

gives

should

which

ATP does not displace

of the

it

PFK catalyzed

of the kinetic but,

by interfering

(191,

as indicated

respect

conditions

case

substrate

ATP acts

the binding

interpretarion

in the

two functions

in an increase

may suppress

This

enzyme.

of the

explanation

librium

significant

(12)

equilibrium

these

absence

active

of subunits

In addition, that

ATP. A possible

resulting

two types

as a normal

dllosteric

of the maximum rate It

in that

functions

of

as inhibitor

KC 5 relationship

concerted

the regulation

does have two distinct

PFK which

concentrations protomers

not

significantly

derably smaller than the number of subunits (6 or 8) which Although this may indicate that in yeast PFK (12, 17, 18). transition

The present

of yeast

ATP and S'AMP behave of three

PFK

behaves

modifies

properties

of physiological

in which

of yeast

conditions

but

regulatory

The number

kinetic

situ"

effector,

of the

respectively.

fit

"in

effector

enzyme show that

allosteric

as a "K system",

activator, best

under

features

can be described phosphate

as an allosteric

at the by the

of phosphate. shape

of the

Vol. 78, No. 4, 1977

saturation

curves

the range tic

as already

phosphate

discussed site

analogs

mechanism

for like

6-phosphate.

of glycolytic appear

oscillations

Furthermore, yeast (21) noted,

however,

considerably

that

narrower

has been used until tions

based

the peculiar phosphate

show that on the

correct,

PFK can be correlated that

of the

induces

role

that

into

respect

in our

the model

behavior

from

a qualitative

observation

range

(22)

studies

studies

point

of view.

behavior It

should

PFK appears

case of the enzyme from E. coli in model

yeast

of

of Hess and Boiteux

extracts.

of yeast

laboratory

of PFK (9-11)

on the allosteric in yeast

of

to adenyla-

now in progress

allosteric

with

the allosstudy

In this

the previous

in the

insight

of phosphate,

oscillations

now as reference

the occurence be tested

in the presence

at least

the allosteric

In addition,

regard,

kine-

a more complex

by an exhaustive

is

in

Thus this

PFK should

enzyme with

of phosphate

with

cooperativity

probably

Further

be provided

as a "K system".

to be essentially

(20).

to yeast

Such a study

results

apparently

et al.

or arsenate.

PFK could

high 6-phosphate.

requires

binding

properties

The present PFK behaves

by Freyer

sulfate

binding

and fructose

by fructose

of phosphate

phosphate

of yeast

the equilibrium tes

show at low ATP concentration

in the absence

of a specific teric

which

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of 0 to 20 % of saturation

complexity

model

BIOCHEMICAL

of glycolytic

be to be

(3)

which oscilla-

(9,lO).

ACKNOWLEDGMENTS We are indebted to Professor J. Yon for her interest to this work and for stimulating discussions. We thank D. Thusius for careful reading of the manuscript. This work was supported by grants from the Centre National de la Recherche Scientifique (G.R. no 13).

REFERENCES ---

(1)

(2) (3) (4) (5)

(6) (7)

(8) (9) (10)

Ramaiah, A. (1974) in : Current topics in cellular regulation (Horecker and Stadtman eds.), Vol. 10, pp. 297-345, Acad. Press. Hess, B., Boiteux, A. and Kriiger, J. (1969) in: Enzyme regulation, Vol. 7, pp; 149-167, Pergamon Press. Blangy, D., But, H. and Monod, J. (1968) J. Mol. Biol., 31, 13-35. Lad, P.M., Hill, D.E. and Hammes, G.G. (1973) Biochem. 12, 22, 4303-4309. Ramaiah, A., Hathaway, J.A. and Atkinson, D.E. (1964) J. Biol. Chem. 239, 1, 3619-3622. R.W. and Ghosh, A. (1964) Proc. Natl. Acad. Chance, B., Estabrook, Sci. USA 51, 1244-1251. Hess, R. and Boiteux, A. (1968) Hoppe Seyler's 2. Physiol. Chem. 349, 1567-1574. Selk'ov, E.E. (1968) Eur. J. Biochem. 4, 79-86. Goldbeter, A. and Lefever, R. (1972) Biophys. J. 12, 1302-1315. Boiteux, A., Goldbeter, A. and Hess, B. (1975) Proc. Natl. Acad. Sci. USA 72,3829-3833. 1294

Vol. 78, No. 4, 1977

(11)

(12) (13) (14) (15) (16) (17)

(18) (19)

(20) (21) (22)

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Goldbeter, A. and Nicolis, G. (1976) in : Progress in theoretical biology, Vol. 4, pp. 65-160, Acad. Press. Diezel, W., EZhme, H.J., Nissler, K., Freyer, R., Heilman, W., Kopperschlgger, G. and Hofmann, E. (1973) Eur. J. Biochem. 38, 479-488. Atkins, G.L. (1971) Biochim. Biophys. Acta 252, 405-420. But, M.H. and But, H. (1967) in : Regulation of enzyme activity and allosteric interactions (Kvamme and Pihl eds.), pp. 109-130, Acad. Press. Monod, J., Wyman, J. and Changeux, J.P. (1965) J. Mol. Biol. 12, 88-118. Banuelos, M., Mazon, M.J., Gancedo, J.M. and Gancedo, C. (1975) 10th FEBS Meeting abs., 912. Tamaki, N. and Hess, B. (1975) Hoppe Seyler's 2. Physiol. Chem., 356, 4, 399-415. Kopperschlsger, G., Usbeck, E. and Hofmann, E. (1976) Biochem. Eiophys. Res. Commun. 71, 1, 371-378. But, H., Johannes, K.J. and Hess, B. (1973) J. Mol. Eiol. 76, 199-205. Freyer, R., Eschrich, K. and Schellenberger, W. (1976) Studia Biophysics, 57, 123-128. Hess, B. and Boiteux, A. (1973) in : Biological and Biophysical oscillators (Chance, Pye, Ghosh and Hess eds.), pp. 229-241, Acad. Press. Rubin, M.M. end Changeux, J.P. (1966) J. Mol. Biol. 21, 265-274.

1295